This invention relates to antibodies against vascular permeability factor and, more particularly, to a method of producing said antibodies by immunization with synthetic peptide fragments of a vascular permeability factor.
Vascular permeability factors (VPFs) are proteins originally obtained from a variety of tumors which cause a rapid and reversible increase in blood vessel permeability when nanogram amounts are injected under the skin of a warm blooded mammal. VPF activity has been found in tumor ascites fluids from guinea pigs, hamsters and mice and is secreted by these tumors and a variety of tumor cell lines in vitro according to Senger et al., Science 219, 983-985 (1983).
In U.S. Pat. No. 4,456,550, a purified VPF is described which has the following characteristics:
(a) in an aqueous solution (0.01M Na.sub.3 PO.sub.4, pH 7) whose concentration of NaCl is varied linearly, VPF is eluted from a heparin-Sepharose chromatography column in a peak centered at 0.4 NaCl; PA1 (b) in an aqueous solution of Na.sub.3 PO.sub.4 (pH 7.0) whose concentration is varied linearly, VPF is eluted from a hydroxylapatite column in a peak centered at 0.25M Na.sub.3 PO.sub.4 ; and PA1 (c) when subjected to SDS gel electrophoresis in a 7.5% polyacrylamide slab gel (0.375M tris-HCl, pH 8.8, 0.1% SDS) at 35 milliamps and 4.degree. C., VPF is localized to a region corresponding to a molecular weight between 34,000 and 45,000 daltons. PA1 (a) affinity chromatography with a column of heparin-Sepharose; PA1 (b) chromatography with a column of hydroxylapaptite; and PA1 (c) sodium dodecylsulfate/polyacrylamide gel electrophoresis. PA1 (a) it has a M.sub.r about 34,000-40,000 as determined by sodium dodecylsulfate polyacrylamide gel electrophosphoresis (SDS/PAGE); PA1 (b) it is a disulfide-linked protein dimer; PA1 (c) it has a N-terminal amino acid sequence as follows: ##STR2## (d) it exhibits substantial mitogenic activity to endothelial cells in culture. PA1 (a) affinity chromatography of said conditioned culture medium with a column of heparin-Sepharose CL-6B; PA1 (b) cation exchange chromatography of the VPF active fractions from said affinity chromatography with a TSK SP-5-PW column; PA1 (c) high performance liquid chromatography (HPLC) of the VPF active fractions from said cation exchange chromatography with a Vydac C.sub.4 reversed phase HPLC column; and PA1 (d) HPLC of the VPF active fractions from said C.sub.4 HPLC with a Vydac C.sub.18 reversed phase HPLC column.
The VPF was purified about 1800 fold from serum-free conditioned medium of guinea pig tumor cell culture or 10,000 fold from ascites fluid by a series of steps consisting of:
As little as 200 ng (5.times.10.sup.-12 moles) of this purified VPF increased the vascular permeability equivalent to 1.25 .mu.g (4.times.10.sup.-9 moles) of histamine. Histamine is a standard permeability mediator described by Miles and Miles, J. Physiol. 118, 228-257 (1952). The VPF is said to have therapeutic value insofar as it enables blood nutrients to reach tissue with increased need for nutrients, as in wound healing. Antibody against the VPF is said to have therapeutic value insofar as it blocks a tumor's ability to increase vessel permeability and thereby to obtain nutrients from increased vessel leakage.
According to Folkman and Klagsbrun, Science 235, 442-447 (1987), VPF causes leakage of proteins, including fibrinogen, from blood vessels, thereby initiating the formation of a fibrin gel which, in turn, may play a role in angiogenesis. See also Dvorak et al., J. Immunol. 122(1), 166-174 (1979); Dvorak, N. Engl. J. Med. 315, 1650-1659 (1986); Kadish et al., Tissue Cell 11, 99, (1979); Dvorak et al., J. Natl. Cancer Inst. 62, 1459-1472 (1979); and Senger et al., Cancer Res. 46, 5629-5632 (1986).
Lobb et al., Int. J. Cancer 36, 473-478 (1985), partially purified a VPF from a human adenocarcinoma cell line HT-29 having a molecular weight of 45,000. This VPF, however, does not bind to immobilized heparin as does the VPF derived from guinea pig tumor cell material by Senger and Dvorak.
The copending application Ser. No. 87,739, filed Aug. 31, 1987, a method of stimulating endothelial cell growth is provided which comprises subjecting said cells to a growth stimulating amount of a highly purified vascular permeability factor (VPF). The disclosure of said copending application is incorporated herein by reference. The highly purified VPF has the following characteristics:
The foregoing highly purified VPF was isolated from serum-free conditioned culture medium of guinea pig tumor cells in a series of steps comprising:
The mitogenic activity of the VPF to endothelial cells in culture was demonstrated by an increase in cell number and in .sup.3 H-thymidine incorporation into deoxyribonucleic acid (DNA) relative to control cultures after several days following addition of the VPF to cultures of bovine aortic endothelial (BAE) cells. In these tests, half maximal stimulation of growth was observed at 5.times.10.sup.-11 M VPF, and half maximal stimulation of .sup.3 H-thymidine incorporation into DNA was observed at 2.5.times.10.sup.-12 M VPF. The in vitro mitogenic response, as measured by the .sup.3 H-thymidine incorporation, was thus about 400 times more sensitive to VPF concentration than the in vivo permeability response since the dose required for a measurable increase in permeability is 1.times.10.sup.-9 M. The mitogenic activity of VPF to endothelial cells in culture thereby is distinct and separate from its vascular permeability activity and was surprising and unexpected since the VPF did not stimulate or only slightly stimulated .sup.3 H-thymidine incorporation or cell growth of other cell types such as 3T3 mouse fibroblasts and mouse smooth muscle cells.